Correlating the differences in the receptor binding domain of SARS-CoV-2 spike variants on their interactions with human ACE2 receptor

Spike glycoprotein of SARS-CoV-2 variants plays a critical role in infection and transmission through its interaction with human angiotensin converting enzyme 2 (hACE2) receptors. Prior findings using molecular docking and biomolecular studies reported varied findings on the difference in the interactions among the spike variants with the hACE2 receptors. Hence, it is a prerequisite to understand these interactions in a more precise manner. To this end, firstly, we performed ELISA with trimeric spike glycoproteins of SARS-CoV-2 variants including Wuhan Hu-1(Wild), Delta, C.1.2 and Omicron. Further, to study the interactions in a more specific manner by mimicking the natural infection, we developed hACE2 receptors expressing HEK-293T cell line, evaluated their binding efficiencies and competitive binding of spike variants with D614G spike pseudotyped virus. In line with the existing findings, we observed that Omicron had higher binding efficiency compared to Delta in both ELISA and Cellular models. Intriguingly, we found that cellular models could differentiate the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2 receptors. Our study using the cellular model provides a precise method to evaluate the binding interactions between spike sub-lineages to hACE2 receptors.

www.nature.com/scientificreports/ coding mutations) in spike protein compared to other VOCs. Due to the higher transmission rate, Omicron has spread rapidly and become the dominant circulating variant globally and has replaced all other previous VOCs 6,7 . Hence, it is critical to understand the binding efficiencies of these variants to the hACE2 receptors to understand potencies of infectivity and transmission rates particularly of dominant Delta and Omicron variants. Multiple computational and experimental studies were attempted to understand the impact of the mutations on binding of Delta and Omicron to hACE2 receptors. However, there were some discrepancies among these studies on evaluating the binding efficiencies of these variants. Several computational studies predicted that the RBD of Omicron showed stronger binding to hACE2 receptors compared to RBD of Wild and Delta respectively [8][9][10][11][12][13][14] . Leyun Wu et al. 15 reported that RBD of Omicron had a weak affinity to hACE2 receptors compared to RBD of Delta, but had similar affinity as RBD of Wild by molecular dynamics (MD) simulations analysis. Similar findings were observed with ELISA, in which RBD of Delta showed higher binding than RBD of Omicron 15,16 . Several attempts were made using multiple biomolecular interaction techniques such as microscale thermophoresis (MST), bio-layer interferometry (BLI), Surface Plasmon Resonance (SPR) to understand affinity between spike variants and hACE2 receptors. Seonghan Kim et al. demonstrated that the RBD of Omicron has a higher affinity towards hACE2 than RBD of Delta by MST analysis. Contrasting results were found in the Maren Schubert et al. study 10,16 . However, BLI approach revealed that both RBDs of Omicron and Delta bind with similar affinities towards hACE2 17 . The inconsistency in evaluating the binding affinities were also observed with SPR analysis as well [18][19][20][21][22] . Hence, a comprehensive strategy including comparing multiple techniques is a prerequisite for a precise understanding of spike variant-hACE2 interactions. Towards understanding the differences in the binding affinities of spike variants including Wild, Delta, Omicron and C.1.2 with hACE2 receptors, in this study, we employed three different methods including ELISA, in vitro flow cytometric binding and competitive binding assay. We also followed three parameters (i) To mimic natural forms of spike protein on SARS-CoV-2 variants, we used trimeric form of spike variants (mutational profile of each variant given in Fig. 1) rather than RBD domain to assess the binding activity towards hACE2 receptors (ii) The differential mutational profiles of spike variants affect the molecular weight of each spike protein of variants. For homogeneity, we evaluated binding efficiencies in molarity, (iii) Binding efficiency of individual spike variants is compared between soluble form (recombinant protein) as well as dimeric (expressed on the cell surface) form of hACE2 receptors.

Results
Binding efficiency of trimeric spike variants to soluble hACE2 receptors. Mutation profiles of the spike variants in their native trimeric form affects the proportions of closed to open confirmations of RBD that in turn affect their binding affinities with hACE2 receptors. Wild strain showed more 'all RBD closed confirma- Figure 1. 3D crystal structure of ancestral SARS-CoV-2 spike trimer (PDB ID 6XR8) 23  www.nature.com/scientificreports/ tion' (reduces the affinity) than the other VOC, whereas, Omicron, highly evolved variant showed almost 100% RBD open confirmations 5,24 . Hence, it is important to evaluate the binding affinities of the variants in their native trimeric forms. To this end, firstly, we evaluated the binding efficiencies of trimeric spike variants towards soluble hACE2 receptors using ELISA assay ( Fig. 2A). We used non-competitive ELISA for titrating different concentrations of each variant with soluble hACE2 receptors. We calculated the EC 50 by concentration-response curves for binding of SARS-CoV-2 spike variants to hACE2 using % of maximum ligand binding values. The data showed that Omicron had 6-fold stronger binding towards hACE2 receptors (EC 50 -0.38 nM), than Delta (EC 50 -0.48 nM, 4.8-fold) and C.1.2 (EC 50 -0.61 nM, 3.7-fold) when compared to spike protein of Wild (Fig. 2B). From these findings, Omicron showed superior binding efficiencies towards the hACE2 receptors when compared to Delta and C.1.2 variants.
Evaluating the binding efficiencies between spike variants using hACE2 receptors expressing cell line. To closely mimic the natural infection, hACE2 receptors were expressed on the surface of HEK-293T cells and evaluated the binding efficiency of these variants using in vitro flow cytometry binding assay. Firstly, the hACE2 gene was cloned into lenti-backbone and produced lentiviral particles encoded the hACE2 receptors (Fig. S1). These particles were transduced into HEK-293T cells for the generation of stable hACE2 overexpressing cells (293T-hACE2). The expression of hACE2 on 293T-hACE2 cells was confirmed by qPCR ( Fig. 3A), which showed overexpression of hACE2 mRNA (~ 7000-fold) and western blot (Figs. 3Band S4), respectively. Further, we confirmed the surface expression of hACE2 receptors on the cells by flow cytometry and fluorescent confocal microscopy ( Fig. 3C-F).
Next, we performed in vitro flow cytometry binding assay at different concentrations of trimeric spike variants ( Fig. 4A) in 293T-hACE2 cells. Although the percentage of spike trimer binding to hACE2 receptors on the cells was similar among Wild, Omicron, Delta, and C.1.2 spike-stained cells (Figs. 4B and S2), the amounts of spike trimer bound per 293T-hACE2 cells (MFI value) were higher in Omicron, Delta, and C.1.2 spike-stained cells compared to Wild spike-stained cells (Figs. 4C and S3). The fold change of spike binding was calculated by normalizing to Wild spike. We found that around 1.3-2.5-fold increase in their binding efficiencies at higher concentrations of compared to Wild (Fig. 4D). Further, we analysed the EC 50 by concentration-response curves using % of maximum ligand binding values between Delta, Omicron and C.1.2 spike trimers. The data showed Omicron spike had stronger binding to hACE2 on the surface of the cells (EC 50 -0.78 nM) than Delta (EC 50 -1.2 nM) and C.1.2 spike (EC 50 -1.1 nM), (Fig. 4E). These findings indicate that Omicron shows stronger binding to hACE2 receptors in homodimeric form (expressed on the cells). Moreover, this data revealed that binding efficiencies of both Delta and C.1.2 were similar to hACE2 receptors in its native form on the cells.

Competing of spike pseudovirus with spike variants, differentiate closely related variants.
To confirm further, we analysed binding efficiencies of spike variants in the presence of D614G spike pseudovirus at different concentrations in 293T-hACE2 cells (Fig. 5A). The pseudovirus has dual reporter genes, ZsGreen for visualization and luciferase for quantification of the infection. Binding of spike variants to the receptor, inhibits the infection of pseudovirus, in turn reduces the expression of reporter genes. Inhibitory concentration (IC) of each spike variant is calculated by the luciferase expression from the pseudovirus infection, in the presence of respective variant. Firstly, inhibition of D614G spike pseudovirus (ZsGreen expression) was visualised in fluorescent microscopy. We observed that Omicron, Delta, and C.1.2 spike proteins inhibited the D614G spike pseudovirus infection more efficiently compared to Wild spike protein in 293T-hACE2 cells (Fig. 5B). Next, we evaluated luciferase expression for highly sensitive detection. Consistent with above two methods, spike protein of Omicron showed lower IC 50 -1.1 nM, compared to Delta (IC 50 -1.4 nM), C.1.2 (1.3 nM) and Wild strain (IC 50 -7 nM) ( Fig. 5C-F). Which indicated that Omicron could effectively inhibit the pseudovirus infection at the lowest concentration compared to the other variants and correlated to the superior binding affinity to the hACE2 receptors.The receptor affinity was increased to spike of Omicron (6.2-fold), Delta (5.1-fold), and C.1.2 (5.4-fold) when compared to Wild spike (Fig. 5G). Since mild differences in the affinities of Delta and C.1.2 spike   Spike variants showed high immune evasion properties against monoclonal antibody. Our findings in both ELISA-based assays and cellular models collectively demonstrated that mutational profile on the RBD domain of spike variants increased their binding towards the hACE2 receptors. Spike affinity to hACE2 receptors and immune evasion are responsible for circulation of new variants in human populations. Hence, we also analysed whether the mutations on RBD domain of spike variants affect its antibody binding. To this end, we screened anti-RBD mAbs (raised against the RBD domain of Wild spike) binding to spike variants by ELISA (Fig. 6A). We observed that anti-RBD mAbs showed higher affinity to Wild (EC 50 -0.13 µg/ml), but affinity was significantly reduced to Delta (EC 50 -0.66 µg/ml), Omicron (EC 50 -0.52 µg/ml) and C.1.2 (EC 50 -0.54 µg/ml) variants (Fig. 6B). This study revealed that mutations on spike protein of Delta, C.1.2 and Omicron resulted in  www.nature.com/scientificreports/ immune evasion. Collectively, these studies revealed that Omicron had higher hACE2 receptors binding affinity with immune evasion, resulting in sustaining the current global prevalence.

Discussion
Understanding the interaction between spike protein variants and the hACE2 receptors is critical for evaluating the infectivity and transmission rates of the VOCs. Most of the studies including molecular modelling studies, biomolecular interaction studies and ELISA were confined to the RBD domain, not to the stable homodimer form of the hACE2 receptors [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . Evaluating the binding energies of trimeric spike to the native form of receptor using molecular modelling simulations is limited by its large size 25 . SPR and BLI-based method involve chemical modifications on the biomolecules for attaching onto the surface of the chip 15 . Recent studies have shown inconsistent data when using certain techniques to analyse the receptor-binding domain (RBD) of spike proteins. This suggests that chemical modifications of the RBD may have an impact on its binding affinity to the receptor and its ability to form a trimer. As a result, these modifications are likely to affect the structure and function of the spike protein differently than the non-modified RBD. Therefore, it is expected that these chemical modifications could lead to discrepancies in binding affinity and spike trimer function. This highlights the need for further exploration of the potential effects of these modifications on the spike protein. Considering the limitations in these techniques, we compared 3 different techniques for evaluation of binding efficiencies. To this end, firstly, we employed non-competitive ELISA, coated with soluble hACE2 receptors, and evaluated the binding efficiencies of trimeric spike from Wild, Delta, C.1.2 and Omicron variants. The trimeric spike of omicron has a 6-fold highest affinity toward soluble hACE2 receptors compared to Delta (4.8-fold), C.1.2 (3.7-fold) than Wild (Fig. 2). More importantly, recombinantly produced soluble hACE2 (1-740 amino acid residues) are less stable, and spontaneously converted into monomeric form in in vitro condition, hence it needs to be stabilized with dimeric FC domain 26,27 . Moreover, recent findings reported that dimeric forms of hACE2 either in soluble or on the cell surface enhanced the spike protein binding than monomeric forms and disruption in the dimerization significantly affected the interactions [26][27][28][29] . Hence, it is ideal to evaluate the receptor binding interactions of spike variants in a close-to-natural phenomenon.
Evaluating and correlating the binding efficiencies of spike variants in cellular models is still an elusive. To this end, we studied the binding efficiencies of spike proteins with hACE2 receptors, expressed as homodimeric form on the surface of the cells using biotinylated trimeric spike variants followed by fluorescently conjugated streptavidin binding and evaluated the fluorescent intensity in flow cytometry. Consistence with ELISA using soluble hACE2 receptors data, the affinity of trimeric spike of Omicron showed higher affinity towards hACE2 receptors on the cells than Delta and C.1.2., but the affinity of a trimeric spike of C.1.2 variants showed slightly higher than Delta variant towards hACE2 receptors on the cell surface (Fig. 4E). We further compared these interactions in a competitive binding experiment by evaluating the levels of infectivity of SARS-CoV-2 D614G spike pseudovirus in presence of trimeric spike variants. D614G is the first single point mutation in spike glycoprotein that contributes to an increased binding of SARS-CoV-2 virus to hACE2 receptors and results in higher rates of infectivity 30 . All the variants including Delta, C.1.2., and Omicron are evolved from D614G lineage (Fig. 1). Hence, we used D614G spike pseudovirus for studying the binding interactions. Competitive binding with the pseudovirus revealed the trimeric spike of Omicron (3-fold) and C.1.2 (2.5-fold) variant was found to be higher affinity to hACE2 receptor when compared to Delta variant (Fig. 5G).
Both computational studies and in vitro studies demonstrated that N501Y mutation on spike protein enhanced the binding affinity towards hACE2 receptors [31][32][33][34] . In concurrence, we observed similar binding affinity of N501Y mutation containing variants including Omicron and C.1.2 showed increased binding to hACE2 receptors than Delta (does not containing N501Y mutations) in the cellular model. Rapidly expanding variant (Omicron) showed higher affinity than the C.1.2., possibly because of additional mutations such as S477N and Q498R in Omicron variants, known to strengthen the affinity of hACE2 as well as cross-species ACE2 receptors 32 .
This study provides a precise approach for evaluating the binding strength of spike to cognate receptors. Using two different approaches involving cellular models, we identified the subtle differences between the closely related C.1.2 and Delta in their binding to hACE2. Overall, this approach would be helpful in evaluating the binding efficiencies of emerging variants to understand the virus-host interactions, disease progression and in screening of potent therapeutic molecules.

Materials and methods
Cloning of hACE2 gene into lentiviral transfer plasmid. The hACE2 ORF sequence amplified from the hACE2 vector (Addgene, #1786) by high fidelity Q5 polymerase PCR using primer set (Table S1) and cloned purified hACE2 gene fragment into NheI and BamHI digested fragment of pLenti backbone (Addgene, #112675) by Gibson assembly. The hACE2 clones (pLenti-hACE-P2A-Puro) were confirmed by PCR and restriction digestion with NheI and BamHI enzymes.
qRT-PCR analysis. We used the protocol for qRT-PCR as described 35 . Total RNA was isolated from one million cells using RNA iso-plus reagent as per manufacture protocol (Takara) from HEK-293T and 293T-hACE2 cells. 500 ng of total RNAs are converted into cDNA using cDNA synthesis kit (Takara). Syber green-based qPCR was performed using primer sets (Table S1)  In vitro flow cytometry binding assay. 1

Statistical analysis.
The statistical significance and visualization of data were generated in GraphPad Prism version 8.0. Statistical significance of spike variant binding to hACE2 receptors was analysed by unpaired t-test with Welch's correction. Equal or less than p values, of *p < 0.05, **p < 0.01, considered as significant and ns = non-significant.

Data availability
Original data is included in the article. Additional data can be accessed from the corresponding author on the request. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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